Circadian clocks are endogenous protein machines that integrate external time cues and internal metabolic states to regulate daily rhythms in physiology and behavior in organisms from all kingdoms of life. In the natural world, environmental zeitgebers enable the animal circadian clock to control timing of food intake. Nutrient influx can therefore provide metabolic signals to reinforce environmental signals, promoting synchrony in circadian physiology to balance metabolism and energy use. Initial efforts to dissect the underpinnings of the circadian oscillator and its control over rhythms of life focused on regulation at the transcriptional level, as the core oscillator proteins are transcription factors that collaborate to govern rhythmic expression of genes involved in diverse cellular processes. More recent studies have uncovered complementary non-transcriptional mechanisms, including protein post-translational modifications (PTMs), that are critical for circadian timekeeping. The overall goal of this project is to understand the mechanisms by which metabolic and environmental signals integrate at the post-translational level to regulate circadian physiology, and more importantly the consequences when these signals that have evolved to cooperate are in conflict. We will use the diurnal Drosophila model to test the central hypothesis that nutrient influx through clock-controlled feeding activity regulates the interplay between phosphorylation and O-linked N-Acetylglucosaminylation (O-GlcNAcylation) of cellular proteins to modulate time-of-day specific functions. Protein O-GlcNAcylation is highly sensitive to metabolic input and may play a dominant role in extensive remodeling of cellular protein functions, bypassing changes in gene expression. In Aim 1, we will use time-restricted feeding (TRF) in combination with targeted metabolomics and chemoenzymatic O-GlcNAc labeling to establish the relationships between feeding-fasting cycle, nutrient influx, and O-GlcNAcylation status of cellular proteins. In Aim 2, we will identify cellular proteins that exhibit daily interplay between O-GlcNAcylation and phosphorylation using label-free proteomic approaches. In Aim 3, we will characterize the function of clock protein O-GlcNAcylation events by utilizing tried-and-true molecular and Drosophila behavioral assays. By addressing the 3 questions: When, What, and Why, we will advance our understanding on metabolic regulation of circadian physiology via post-translational mechanisms. This project will have broad significance as cross-talk between protein phosphorylation and O-GlcNAcylation is extensive and modulates a wide range of cellular processes. Our findings may identify new therapeutic targets to alleviate circadian and metabolic disorders.